Defective platelet function in Niemann-Pick disease type C1.

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in either NPC1 (95% of cases) or NPC2. Reduced late endosome/lysosome calcium (Ca2+) levels and the accumulation of unesterified cholesterol and sphingolipids within the late endocytic system characterize this disease. We previously reported impaired lysosome-related organelle (LRO) function in Npc1 -/- Natural Killer cells; however, the potential contribution of impaired acid compartment Ca2+ flux and LRO function in other cell types has not been determined. Here, we investigated LRO function in NPC1 disease platelets. We found elevated numbers of circulating platelets, impaired platelet aggregation and prolonged bleeding times in a murine model of NPC1 disease. Electron microscopy revealed abnormal ultrastructure in murine platelets, consistent with that seen in a U18666A (pharmacological inhibitor of NPC1) treated megakaryocyte cell line (MEG-01) exhibiting lipid storage and acidic compartment Ca2+ flux defects. Furthermore, platelets from NPC1 patients across different ages were found to cluster at the lower end of the normal range when platelet numbers were measured and had platelet volumes that were clustered at the top of the normal range. Taken together, these findings highlight the role of acid compartment Ca2+ flux in the function of platelet LROs.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

BACKGROUND: Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. METHODS: The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. RESULTS: Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. CONCLUSION: These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic ß Cells.

Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.

Absence of intracellular ion channels TPC1 and TPC2 leads to mature-onset obesity in male mice, due to impaired lipid availability for thermogenesis in brown adipose tissue.

Intracellular calcium-permeable channels have been implicated in thermogenic function of murine brown and brite/beige adipocytes, respectively transient receptor potential melastin-8 and transient receptor potential vanilloid-4. Because the endo-lysosomal two-pore channels (TPCs) have also been ascribed with metabolic functionality, we studied the effect of simultaneously knocking out TPC1 and TPC2 on body composition and energy balance in male mice fed a chow diet. Compared with wild-type mice, TPC1 and TPC2 double knockout (Tpcn1/2(-/-)) animals had a higher respiratory quotient and became obese between 6 and 9 months of age. Although food intake was unaltered, interscapular brown adipose tissue (BAT) maximal temperature and lean-mass adjusted oxygen consumption were lower in Tpcn1/2(-/-) than in wild type mice. Phosphorylated hormone-sensitive lipase expression, lipid density and expression of ß-adrenergic receptors were also lower in Tpcn1/2(-/-) BAT, whereas mitochondrial respiratory chain function and uncoupling protein-1 expression remained intact. We conclude that Tpcn1/2(-/-) mice show mature-onset obesity due to reduced lipid availability and use, and a defect in ß-adrenergic receptor signaling, leading to impaired thermogenic activity, in BAT.

TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2.

Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. Two-pore channels (TPCs) are cation channels that reside in endo-lysosomal organelles, and overexpression results in endo-lysosomal trafficking defects. However, the impact of a lack of TPC expression on endo-lysosomal trafficking is unknown. Here, we characterize Tpcn1 expression in two transgenic mouse lines (Tpcn1(XG716) and Tpcn1(T159)) and show expression of a novel evolutionarily conserved Tpcn1B transcript from an alternative promoter, raising important questions regarding the status of Tpcn1 expression in mice recently described to be Tpcn1 knockouts. We show that the transgenic Tpcn1(T159) line lacks expression of both Tpcn1 isoforms in all tissues analyzed. Using mouse embryonic fibroblasts (MEFs) from Tpcn1(-/-) and Tpcn2(-/-) animals, we show that a lack of Tpcn1 or Tpcn2 expression has no significant impact on resting endo-lysosomal pH or morphology. However, differential effects in endo-lysosomal function were observed upon the loss of Tpcn1 or Tpcn2 expression; thus, while Tpcn1(-/-) MEFs have impaired trafficking of cholera toxin from the plasma membrane to the Golgi apparatus, Tpcn2(-/-) MEFs show slower kinetics of ligand-induced platelet-derived growth factor receptor ß (PDGFRß) degradation, which is dependent on trafficking to lysosomes. Our findings indicate that TPC1 and TPC2 have important but distinct roles in the endo-lysosomal pathway.

Presynaptic NMDARs in the hippocampus facilitate transmitter release at theta frequency.

A rise in [Ca(2+)](i) provides the trigger for neurotransmitter release at neuronal boutons. We have used confocal microscopy and Ca(2+) sensitive dyes to directly measure the action potential-evoked [Ca(2+)](i) in the boutons of Schaffer collaterals. This reveals that the trial-by-trial amplitude of the evoked Ca(2+) transient is bimodally distributed. We demonstrate that "large" Ca(2+) transients occur when presynaptic NMDA receptors are activated following transmitter release. Presynaptic NMDA receptor activation proves critical in producing facilitation of transmission at theta frequencies. Because large Ca(2+) transients "report" transmitter release, their frequency on a trial-by-trial basis can be used to estimate the probability of release, p(r). We use this novel estimator to show that p(r) increases following the induction of long-term potentiation.

Dosimetric characteristics of dual-layer multileaf collimation for small-field and intensity-modulated radiation therapy applications.

The purpose of the present work was to measure the performance characteristics in the penumbra region and on the leaf-end of an innovative dual-layer micro multileaf collimator (DmMLC). The DmMLC consists of two orthogonal (upper and lower) layers of leaves; a standard MLC consists of one layer. The DmMLC provides unique performance characteristics in smoothing dose undulation, reducing leaf-end transmission, and reducing MLC field dependence of the leaf stepping angle. Two standard MLCs (80-leaf and 120-leaf versions: Varian Medical Systems, Palo Alto, CA), a DmMLC (AccuKnife: Initia Medical Technology, Canton, MA), and a Cerrobend (Cerro Metal Products, Bellefonte, PA) block were used in performance studies involving a triangular field, a cross leaf-end field, and a circular field. Measurements were made with 6-MV X-rays and extended dose range film at a depth of 5 cm in Solid Water (Gammex rmi, Middleton, WI) at a source-axis distance of 100 cm. The field penumbra width measured between the 20% and 80% isodose lines through the MLC-80, MLC-120, DmMLC, and Cerrobend block were 9.0, 5.0, 3.0, and 2.0 mm respectively. The dose undulation amplitude of the 50% isodose line was measured as 5.5, 2.0, and 0.5 mm for the MLC-80, MLC-120, and DmMLC respectively. The planar dose difference between the MLC-80, MLC-120, and DmMLC against Cerrobend block was measured as ranging at +/-52.5%, +/-35.0%, and +/-20.0% respectively. The leaf-end transmission was measured at 22.4% in maximum and 15.4% in average when closing a single layer of the DmMLC, and at 2.4% in maximum and 2.1% in average when closing both layers. The MLC dependence of the leaf stepping angle with the DmMLC ranged from 45 degrees to 90 degrees. The standard MLC leaf stepping angle ranged from 0 degrees to 90 degrees. In conclusion, the dose undulation, leafend transmission, and MLC field dependence of the leaf stepping angle with the DmMLC were remarkably reduced as compared with those of the standard MLCs. And as compared with Cerrobend block, the DmMLC provided very comparable performance in field-edge smoothing and in the shaping of complex fields.